Nuclear and cytoplasmic specific RNA binding proteome enrichment and its changes upon ferroptosis induction

The key role of RNA-binding proteins (RBPs) in posttranscriptional regulation of gene expression is intimately tied to their subcellular localization. Here, we show a subcellular-specific RNA labeling method for efficient enrichment and deep profiling of nuclear and cytoplasmic RBPs. A total of 1221 nuclear RBPs and 1333 cytoplasmic RBPs were enriched and identified using nuclear/cytoplasm targeting enrichment probes, representing an increase of 54.4% and 85.7% compared with previous reports. The probes were further applied in the omics-level investigation of subcellular-specific RBP-RNA interactions upon ferroptosis induction. Interestingly, large-scale RBPs display enhanced interaction with RNAs in nucleus but reduced association with RNAs in cytoplasm during ferroptosis process. Furthermore, we discovered dozens of nucleoplasmic translocation candidate RBPs upon ferroptosis induction and validated representative ones by immunofluorescence imaging. The enrichment of Tricarboxylic acid cycle in the translocation candidate RBPs may provide insights for investigating their possible roles in ferroptosis induced metabolism dysregulation.


Figure S5 .
Figure S5.Detailed experimental design of the quantitative differential proteomic comparison between the experimental group and control group for RBP identification.

Figure S6 .
Figure S6.Scatter plot for RBPs identification by quantitative differential proteomic comparison.a-f Scatter plot of (a) BLF, (b) BLTF, (c) BL3F, (d) BETF, (e) BELF, (f) BLEF displaying the log2 fold change (x-axis) and -log P values (y-axis) for RBPs identification by quantitative differential proteomic comparison between the experiment group and control group.Red dots represented the identified RBPs with a stringent screening cut-off.Statistical analysis was performed with two-sided Student's t-test (Benjamini&Hochberg (BH) adjusted P values) from three biological replicates.

Figure
Figure S8.RNA-seq quality control metrics by reads counting.Intronic regions are enriched in the nuclear RNA isolated by BLTF.Intergenic regions accounted for only about 5% and 1% of reads in the enrichment result of BLTF and BETF, indicating relatively low levels of DNA contamination.Three biological replicates were repeated with similar results.

Figure S9 .
Figure S9.Validation of subcellular fractionation by western blotting.Lamin A/C and histone 3 were used as nuclear markers and β-tubulin was used as the cytoplasmic maker.

Figure S10 .
Figure S10.Biocompatibility of Hela cells treated with ferroptosis inducers.Biocompatibility of Hela cells after treating with (a) erastin, (b) RSL3 and (c) DAT of different concentrations and incubation times.The cell survival rate was analyzed by the CCK-8 assay.d Ferroptotic cell death measured by flow cytometry of the cells.

Figure S11 .
Figure S11.Confocal fluorescent imaging of probes with and without ferroptosis inducer treatment.a, b The (a) BLTF and (b) BETF probes were stained with FITC (green) via biotin and streptavidin coupling.Hoechst 33342 (blue) was used as a nuclear marker and Mitotracker (red) was used as a mitochondria maker.Scale bars, 20 µm.

Figure S12 .
Figure S12.Quantitative analysis of nuclear/cytoplasmic proteins in cells treated with or without ferroptosis inducer.a-c Volcano plot displaying log2 fold change (FC) (x-axis) and -log10 P values (y-axis) of the nuclear proteome of the (a) erastin-, (b) RSL3-, (c) DAT-treated and nontreated cells.Proteins with significant increase after treatment were depicted in red, and those significantly decreased after erastin treatment were depicted in blue.d-f Volcano plot displaying log2 fold change (FC) (x-axis) and -log10 P values (y-axis) of the cytoplasmic proteome of the (d) erastin-, (e) RSL3-, (f) DAT-treated and nontreated cells.Proteins with significant increase after treatment were depicted in red, and those significantly decreased after erastin treatment were depicted in blue.Statistical analysis was performed with two-sided Student's t-test (BH adjusted P values) from three biological replicates.

Figure S13 .
Figure S13.Quantitative analysis of RBPs in nuclear/cytoplasmic RNPs in cells treated with or without ferroptosis inducers.a, b Volcano plots displaying log2 fold change (FC) (x-axis) and -log P values (y-axis) of the RBPs quantified from the erastin-treated and nontreated cells by (a) nucleustargeting probe BLTF and (b) cytoplasm-targeting probe BETF enrichment.c, d Volcano plots displaying log2 fold change (FC) (x-axis) and -log P values (y-axis) of the RBPs quantified from the RSL3-treated and nontreated cells by (c) nucleustargeting probe BLTF and (d) cytoplasm-targeting probe BETF enrichment.e, f Volcano plots displaying log2 fold change (FC) (x-axis) and -log P values (y-axis) of the RBPs quantified from the DAT-treated and nontreated cells by (e) nucleustargeting probe BLTF and (f) cytoplasm-targeting probe BETF enrichment.Significantly increased RBPs after ferroptosis inducer treatment were depicted in red, and significantly decreased RBPs after ferroptosis inducer treatment were depicted in blue.Statistical analysis was performed with two-sided Student's t-test (BH adjusted P values) from three biological replicates.

Figure S14 .
Figure S14.Overlaps of the ferroptosis inducers regulated subcellular RBPs.a, b Overlap of (a) the upregulated RBPs in nuclear RNPs and (b) the downregulated RBPs in cytoplasmic RNPs induced by erastin, RSL3 and DAT.c Overlap of nuclear upregulation and cytoplasmic downregulation RBPs induced by erastin.

Figure S15 .
Figure S15.Analysis of the erastin-induced nucleoplasmic translocation candidate RBPs. a Pathway analysis of the candidates.b GO biological process analysis of the candidates.Statistical analysis was performed with hypergeometric test (BH adjusted P values).

Figure S16 .
Figure S16.Erastin induced RBPs translocation negative control.a, b Confocal fluorescent imaging of (a) RPL7A and (b) RPS27A as negative control for RBPs with/without erastin induction.c The nuclear/cytoplasmic fluorescence intensity ratio of RPL7A and RPS27A in (a) and (b).Values are the mean ± S.D. of n=10 cells per condition, two-way ANOVA.

Figure S17 .
Figure S17.Quantitative analysis of nuclear proteins in cells treated with or without erastin.Volcano plot displaying log2 fold change (FC) (x-axis) and -log10 P values (y-axis) of the nuclear proteome of the erastin-treated and nontreated cells.Proteins with significant increase after erastin treatment were depicted in red, and those significantly decreased after erastin treatment were depicted in blue.Statistical analysis was performed with two-sided Student's t-test (BH adjusted P values) from three biological replicates.

Figure S18 .
Figure S18.Immunofluorescence imaging of TCA proteins with and without erastin induction.a-e Immunofluorescence images of (a) ACO2, (b) FH, (c) CS, (d) MDH2 and (e) PDHA1 with and without erastin induction.f The nuclear/cytoplasmic fluorescence intensity ratio of ACO2, FH, CS, MDH2 and PDHA1 with and without erastin induction.Values are the mean ± S.D. of n=10 cells per condition, two-way ANOVA.

Figure S19 .
Figure S19.Quantitative analysis of the BLTF probe enriched nuclear RBPs in cells treated with or without erastin after normalization by the corresponding protein abundance variation.Volcano plot displaying log2 fold change (FC) (x-axis) and -log10 P values (y-axis) of the nuclear RBPs enriched from the erastin-treated and nontreated cells after normalization by the nuclear proteome.Significantly increased nuclear RBPs after erastin treatment were depicted in red, and significantly decreased ones were depicted in blue.Statistical analysis was performed with two-sided Student's t-test (BH adjusted P values) from three biological replicates.

Figure S20 .
Figure S20.Quantitative analysis of the CA-RBPs in cells treated with or without erastin.Volcano plot displaying log2 fold change (FC) (x-axis) and -log10 P values (yaxis) of the CA-RBPs quantified from the erastin-treated and nontreated cells.CA-RBPs with significant increase after erastin treatment were depicted in red, and significantly decreased after erastin treatment were depicted in blue.Statistical analysis was performed with two-sided Student's t-test (BH adjusted P values) from three biological replicates.

Table S1 .
List of theoretical and measured molecular weights of the synthesized probes.

Table S2 .
Quantitative analysis of RPS27A and RPL7A identified by subcellular proteome profiling and RNA binding profiling before and after erastin treatment.